Silk‐Gel Powered Adenoviral Vector Enables Robust Genome Editing of PD‐L1 to Augment Immunotherapy across Multiple Tumor Models

Abstract Immune checkpoint blockade based on antibodies has shown great clinical success in patients, but the transitory working manner leads to restricted therapeutic benefits. Herein, a genetically engineered adenovirus is developed as the vector to deliver CRISPR/Cas9 (sgCas9‐AdV) to achieve permanent PD‐L1 gene editing with efficiency up to 78.7% exemplified in Hepa 1‐6 liver cancer cells. Furthermore, the sgCas9‐AdV is loaded into hydrogel made by silk fiber (SgCas9‐AdV/Gel) for in vivo application. The silk‐gel not only promotes local retention of sgCas9‐AdV in tumor tissue, but also masks them from host immune system, thus ensuring effectively gene transduction over 9 days. Bearing these advantages, the sgCas9‐AdV/Gel inhibits Hepa 1‐6 tumor growth with 100% response rate by single‐dose injection, through efficient PD‐L1 disruption to elicit a T cell‐mediated antitumor response. In addition, the sgCas9‐AdV/Gel is also successfully extended into other refractory tumors. In CT26 colon tumor characterized by poor response to anti‐PD‐L1, sgCas9‐AdV/Gel is demonstrated to competent and superior anti‐PD‐L1 antibody to suppress tumor progression. In highly aggressive orthotopic 4T1 mouse breast tumor, such a therapeutic paradigm significantly inhibits primary tumor growth and induces a durable immune response against tumor relapse/metastasis. Thus, this study provides an attractive and universal strategy for immunotherapy.


In vitro adenoviral vector transduction
EGFP fluorescence was used to evaluate the transduction efficiency of sgCas9-AdV. In a typical experiment, the cells (Hepa1-6, SMMC-7721, C3A, SK) were seeded in 6-well plates at a density of 3×10 5 cells per well one day before infection with the sgCa9-AdV (10 7 pfu per well). After 48 h of transduction, the cells were examined for EGFP fluorescence by fluorescence microscopy (Zeiss Axio Vert.A1, Germany).

Animals
Six-week-old C57BL/6 and Balb/c mice were ordered from Shanghai China PCR products were digested with T7 Endonuclease I and run on a 1% agarose gel, finally analyzed with an imaging system (ChemiDoc TM MP, Bio-Rad). The indel efficiency was calculated according to the following formula: (ii+iii)/(i+ii+iii) × 100%, where i is the intact band around 1000 bp, ii and iii are cleavage bands around 300 bp and 700 bp, respectively. The gray value of these bands was quantified using Image J. 1 In addition, PCR products of adenovirus-infected cells were subjected to Sanger sequencing.

Western Blot analysis
Cell lysates were prepared in RIPA lysis buffer (Beyotime Biotechnology, China) containing PMSF and a protease inhibitor cocktail (MedChemExpress, China), and the protein content of the generated cell lysates was determined using the BCA protein assay (TransGen Biotech, China). Aliquots containing 20 μg of total protein were loaded on SDS-polyacrylamide gels and transferred to nitrocellulose membranes.
After membranes were blocked with 5% BSA for 1 h, they were probed with anti-PD-L1 antibody at a dilution of 1:1000 and anti-β-actin antibody at a 1:5000 dilution. After washing, the membranes were incubated with the HRP-conjugated secondary antibodies (1:5000) for 1h at room temperature. Antibody binding was detected using an imaging system (ChemiDoc TM MP, Bio-Rad).

Synthesis of sgCas9-AdV/Gel
Silk-gel was obtained according to our previously published works. 2 For loading with sgCa9-AdV, 1 mL of silk fibroin solution (3 wt%) was sonicated by an ultrasonic probe (Scientz-IID, Ningbo Scientz Biotechnology, China) with a duty cycle of 50% for 180 s, and then immediately mixed with 4 mL of adenovirus particles through a syringe, followed by rested at room temperature for gel formation at the adenovirus particles' concentrations of 2×10 10 pfu/mL (sgCa9-AdV). The morphology was monitored by scanning electron microscopy imaging (Nova NanoSEM 230) at an accelerating voltage of 6 kV.

Evaluation of rheological and swelling properties
For evaluating the rheological property, the hydrogel was placed in a dynamic shear rheometer (M301) to perform rheology experiments. The storage modulus (G′) and loss modulus (G′′) of hydrogels were determined at appropriate strain and stress.
For evaluation of swelling property, the silk-gel and sgCas9-AdV/gel were respectively placed in PBS, and the weight of each group was measured once every day. The swelling ratio was calculated by dividing the measured weight by the initial weight of gel.

Sustained release in vitro
The silk-gel containing sgCas9-AdV was placed in a cell strainer with a pore size of 8 μm. Then, the strainer was embedded in a 24-well plate cultured with 1×10 5 of Hepa 1-6 cells. Every 24 hours, the bottom of the strainer was washed 2-3 times with sterile PBS before transferred to another 24-well plate cultured with uninfected 1×10 5 of Hepa1-6 cells in advance. At last, the cells were photographed by a fluorescent microscope to determine viral release.
When tumors reached an average size of 100 mm 3 , the mice were divided into two groups and injected intratumorally with sgCas9-AdV and sgCas9-AdV/Gel, respectively. The amount of virus injected into the two groups of mice was 1×10 9 pfu.

Neutralizing antibody test
Anti-adenovirus antiserum was prepared as follows. C57BL/6 mice were injected with 1×10 9 pfu of adenovirus through tail vein on day 0 and day 20. Seven days later, the animals were sacrificed and the blood was collected through heart puncture. After 15min at room temperature, the blood was centrifuged at 4 °C for serum separation.
The serum was then diluted with culture medium and incubated with sgCas9-AdV or sgCas9-AdV/Gel for 1 h at 37 °C, followed by being transferred into another 24-well plate pre-seeded with Hepa 1-6 cells to monitor the adenovirus infectivity by fluorescence microscope and flow cytometer.
For isolation and analysis of memory T cells, three mice in each group were sacrificed on day 16 after different treatments, and the spleens were dissected.
Splenocytes were passed through a 40 μm filter and isolated by Ficoll-Paque density gradient centrifugation before being stained with the corresponding antibodies and examined by flow cytometry. The antibodies used for flow cytometry are listed below: Anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, and anti-CD44-PE-Cy7.

Immune memory effect for inhibiting tumor lung metastasis
Orthotopic 4T1 tumor-bearing mice received above mentioned treatments. On day 16, residual tumors were surgically removed from the mice, and the wounds were sutured. At the same time, 5×10 5 4T1 cells were injected into mice through the tail vein. After approximately 2 weeks, lung tissue was isolated from mice with different treatments for H&E staining.

RNA sequencing and bioinformatic analysis
The tumor tissues were extracted and quickly frozen into liquid N 2 . Subsequently, total RNA was extracted using Triol reagent kit according to the manufacturer's protocol. After extraction of total RNA, mRNA was further enriched and fragmented, and cDNA was obtained by reverse transcription. Then, transcriptome libraries were further constructed according to the manufacturer's instructions, and sequenced by the Illumina novaseq6000 platform (paired end, 150 bp).
All qualified raw reads were aligned to the mouse genome (mm10, https://www.gencodegenes.org) using STAR, 3 then expression levels of genes were further quantified using FPKM (transcripts per million) value and differential expression analysis between different groups was conducted by Cufflinks v2.2.1, 4 and KEGG pathway enrichment analysis was conducted with clusterProfiler package using differentially expressed genes (At the same time to meet the following two conditions: first. In the two groups of samples, the FPKM is greater than 0.5, or when the FPKM of one sample is 0, the FPKM of the other group should be greater than 1. Secondly, the Fold change is greater than 1.5 or less than 0.6667).

Statistical Analysis
Data were presented as mean ± standard deviation (SD). The data were analyzed by one-way analysis of variance (ANOVA) or t-test as indicated. The log-Rank test was used to compare survival differences. Prism 6 software (GraphPad) was used to perform all statistical analyses. Results were considered statistically significant when *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.           . Statistical significance was calculated by one-way ANOVA test, *P < 0.05, **P < 0.01 and ***P < 0.001. ns means no significant difference.    Figure 4A and B.
To detect the percentage of CD3 + CD4 + and CD3 + CD8 + T cells in the Hepa1-6 tumor. Figure S15. Representative flow cytometry gating strategies for experiments in Figure 4C and D.
To detect the percentage of Treg and IFN-γ + CD8 + T cells in the Hepa1-6 tumor.